Abstract
Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated, gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (ψAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, ψAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera, though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site within GSTE5 may represent adaptations to new habitats.
Highlights
Gene duplications are a major mechanism for acquisition of proteins with novel functions
Introns were small, ranging from 59–75 bp in An. stephensi, 61–83 bp in An. funestus and 60–105 bp in An. plumbeus (Table 1) and can be classified as phase 0 introns, with the exception of the second intron in GSTE7 and the GSTE6 intron which can be classified as phase 1 introns
In this study the order and sequence of the insect specific glutathione S-transferase (GST) epsilon (GSTE) cluster of An. funestus and An. stephensi, both belonging to the Cellia subgenus, and of Anopheles plumbeus, from the Anopheles subgenus, were characterised and compared to those of An. gambiae
Summary
Gene duplications are a major mechanism for acquisition of proteins with novel functions. One group of detoxification associated genes, the Glutathione S-Transferases (GSTs), appears to have undergone multiple independent radiations in the Diptera, e.g. in Drosophila [2] and Lepidoptera (Bombyx mori) [3] This is a marked contrast with hymenopterans where in both Apis [4] and Nasonia [5] there is a relative paucity of GSTs. notable is the insect specific epsilon class (GSTE) in the Culicidae which has apparently undergone independent expansions in Anophelinae and Culicinae subfamilies – whilst both Aedes aegypti and Anopheles gambiae contain eight GSTEs, only four putative orthologs (GSTE2-4 and GSTE8) are identifiable, suggestive of independent gene duplication events [6]. Quantitative genetic studies of a DDT-resistant An. gambiae colony localised a QTL around the GSTE cluster on chromosome 3R [11]
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